Renal cancer is typically silent and frequently fatal. In the United States in 2009 alone over 57,000 new kidney cancers were diagnosed and almost 13, 000 deaths were attributable to this disease. Early diagnosis, triggering surgical excision, is usually curative. Physical signs and symptoms occur late, and by the time renal cancer is consequently diagnosed, it has usually metastasized to lymph nodes or adjacent organs and advanced beyond a curative stage with minimal chance for survival. Incidental early discovery may occur fortuitously during abdominal imaging (CT, MRI, ultrasound) for unrelated causes. Nonetheless, there is presently no method for population screening and diagnosis of renal cancer, particularly at a curative stage. The unmet need is a simple, high through-put, cost-effective test for kidney cancer screening, with the public health significance of preventing over 13,000 deaths annually especially in at risk populations. The proposed research will test the hypothesis that renal cancer cells express and shed or actively secrete proteins into the urine that are signature molecular markers (biomarkers), and that these biomarkers are an early disease indicator, detectable when the tumor is at a treatable stage. [In an in press publication, we have shown that aquaporin 1 (AQP1) and adipophilin (ADFP) are significantly increased in urinary exosomes of patients with kidney cancer thus providing the first sensitive and specific biomarkers of this disease.] Therefore, it is further hypothesized that urine exosomes, vesicular structures released into the urine by all parts of the nephron, will be a [discovery tool for additional novel biomarker proteins and will serve as a non-invasive biopsy of kidney cell oncogenesis and metastasis]. The overall objective is to identify proteins in the urine exosomes of kidney cancer patients which are unique to this disease, and which can be developed into a diagnostic test to screen [whole urine] to detect kidney cancer at an early curable stage and to follow patients post-operatively for recurrence. The specific aims are a discovery stage to: 1) Identify candidate biomarker proteins in the urine exosomes of patients with kidney cancer which are pathogneumonic of the disease; a development stage to 2) Develop sensitive and specific ELISAs to measure levels of novel biomarker proteins identified in Aim1; and a validation stage to 3) Characterize the candidate biomarker proteins to determine their diagnostic sensitivity and specificity. Unique up- or down-regulated proteins will be identified by unbiased top-down and supervised bottom-up mass spectral (LC/MS/MS) and Western blot proteomic analysis of the urine exosomal proteins. Once proteins are identified in training sets of patients by mass spectrometry, precise but economical ELISA-based immunologic assays that give same-day results will be developed, for protein quantification [in whole urine and serum] in validation sets of patients. Overall, our study will improve the diagnosis of kidney cancer to a stage early enough to allow a significant chance for cure while maximizing future kidney function, [follow patients post-operatively for recurrence and identify future targets for selective intervention].